Daidzein Inhibits Human Platelet Activation by Downregulating Thromboxane A2 Production and Granule Release, Regardless of COX-1 Activity

Platelets play crucial roles in cardiovascular diseases (CVDs) by regulating hemostasis and blood coagulation at sites of blood vessel damage. Accumulating evidence indicates daidzein inhibits platelet activation, but the mechanism involved has not been elucidated. Thus, in this study, we investigated the mechanism responsible for the inhibition of collagen-induced platelet aggregation by daidzein. We found that in collagen-induced platelets, daidzein suppressed the production of thromboxane A2 (TXA2), a molecule involved in platelet activation and aggregation, by inhibiting the cytosolic phospholipase A2 (cPLA2) signaling pathway. However, daidzein did not affect cyclooxygenase-1 (COX-1). Furthermore, daidzein attenuated the PI3K/PDK1/Akt/GSK3αβ and MAPK (p38, ERK) signaling pathways, increased the phosphorylation of inositol trisphosphate receptor1 (IP3R1) and vasodilator-stimulated phosphoprotein (VASP), and increased the level of cyclic adenosine monophosphate (cAMP). These results suggest that daidzein inhibits granule release (ATP, serotonin, P-selectin), integrin αIIbβ3 activation, and clot retraction. Taken together, our study demonstrates that daidzein inhibits collagen-induced platelet aggregation and suggests that daidzein has therapeutic potential for the treatment of platelet aggregation-related diseases such as atherosclerosis and thrombosis.


Introductions
Cardiovascular disease (CVD) is a major, growing problem worldwide. Platelet-related cardiovascular disease refers to conditions in which abnormalities in platelet function or excessive platelet activation contribute to the development and progression of cardiovascular disorders. Platelets are small, disc-shaped cells in the blood that play a crucial role in blood clotting. Normal platelets play a critical role in preventing blood loss when blood vessels are damaged. While platelets are necessary for wound healing and preventing excessive bleeding, excessive or abnormal platelet aggregation is a contributory factor in CVDs such as thrombosis, stroke, and atherosclerosis [1][2][3].
Platelets are metabolically active cells that lack a nucleus and exist as small cellular fragments containing numerous functional organelles, including endoplasmic reticulum, Golgi apparatus, and mitochondria [4]. Signaling within platelets occurs after receptors on platelet surfaces are activated by agonists such as collagen, thrombin, and adenosine diphosphate (ADP) [5]. When blood vessels are injured, platelet agonists such as collagen, ADP, and thrombin are exposed or produced locally at sites of injury. In addition, the platelet glycoprotein VI (GPVI) receptor-mediated signaling pathway primarily responds to collagen-induced platelet activation [6,7].
Granule release is a process that occurs in platelets during platelet activation. Platelets contain different types of granules, including dense granules and alpha granules, which store various bioactive molecules [8]. Dense granules release molecules such as serotonin, ADP, calcium, and adenosine triphosphate (ATP). These molecules play a role in promoting platelet aggregation and blood clot formation. They act by activating nearby platelets and enhancing the recruitment of additional platelets to the site of injury [9]. Alpha granules in platelets contain various proteins, one of which is P-selectin. P-selectin is translocated to the platelet surface upon activation. Its interaction with P-selectin glycoprotein ligand-1 facilitates leukocyte adhesion and recruitment, contributing to immune response and inflammation. Additionally, P-selectin is involved in platelet-platelet interactions and platelet aggregation, playing a role in hemostasis and thrombus formation [10,11].
When platelets are activated, cytosolic phospholipase A 2 (cPLA 2 ) releases arachidonic acid (AA) from cell membrane phospholipids. AA can then be converted into various inflammatory mediators by enzymes such as cyclooxygenase-1 (COX-1) [12]. COX-1 catalyzes the conversion of arachidonic acid into prostaglandin H 2 , which is subsequently converted to thromboxane A 2 (TXA 2 ) by thromboxane synthase (TXAS). TXA 2 promotes platelet aggregation and vasoconstriction, contributing to the formation of blood clots [13].
The cyclic adenosine monophosphate (cAMP) signaling pathway influences multiple aspects of platelet function, including the regulation of vasodilator-stimulated phosphoprotein (VASP), inositol trisphosphate receptor (IP 3 R), and integrin activation [14]. IP 3 R is an intracellular calcium channel that is involved in calcium signaling. When platelets are activated by agonists, IP 3 R is stimulated, resulting in the release of calcium ions from intracellular stores. This calcium release is critical for platelet activation and aggregation [15]. On the other hand, phosphorylated VASP has been shown to promote the separation of the α and β subunits of integrin α IIb β 3 , leading to integrin activation and enhanced binding to ligands such as fibrinogen. Integrins are a family of cell surface receptors that mediate cell-cell and cell-extracellular matrix interactions. In platelets, integrin α IIb β 3 is primarily responsible for platelet aggregation by binding to fibrinogen and other ligands [16,17]. Activation of integrin α IIb β 3 is crucial for platelet aggregation and clot formation [18].
When platelets are stimulated by agonists like thrombin or collagen, the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated. PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). PIP 3 acts as a second messenger and recruits Akt to the plasma membrane, where it is activated by phosphorylation [19]. Activated Akt then initiates a cascade of downstream events that contribute to platelet activation. It phosphorylates and activates various targets including proteins involved in granule release and integrin α IIb β 3 activation. These processes promote platelet aggregation, adhesion, and clot formation [20,21].
Isoflavones, which are abundant in soybeans, have garnered considerable attention from the scientific community [22]. Early studies have shown that isoflavones might exert anti-platelet function through cAMP regulation, tyrosine kinase, calcium messenger, and TxA 2 pathway inhibition [23][24][25][26][27]. Genistein, a tyrosine kinase inhibitor, is known to inhibit thromboxane-mediated platelet activation, and daidzein, which has a similar chemical formula, is also known to have anti-platelet effects [25]. Daidzein is a type of isoflavone, a group of compounds found in plants, including soybeans ( Figure 1A). Daidzein has been reported to play a significant role in the prevention and treatment of a variety of diseases such as cancer, CVD, diabetes, osteoporosis, skin disease, and neurodegenerative disease [28]. However, the mechanism responsible for its anti-platelet effects has not been fully elucidated, and thus we investigated this mechanism.  Results are presented as means ± SDs (n = 3). * p < 0.05, ** p < 0.001 versus collagen-treated platelets. NS, not significant versus agonist-treated platelets.

Effects of Daidzein on Human Platelet Aggregation induced by Different Agonists
To confirm the effect of daidzein on human platelet aggregation, we conducted a human platelet aggregation test using PRP and washed platelets. Daidzein (12.5-50 µM) inhibited collagen (2.5 µg/mL)-induced platelet aggregation in a concentration-dependent manner ( Figure 1B) but had no inhibitory effect on thrombin (0.05 U/mL) or ADP (20 µM)induced platelet aggregation ( Figure 1C,D).

Effect of Daidzein on Granule Release by Collagen-Activated Platelets
Activated platelets release alpha granules and dense granules which play important roles in hemostasis [9]. In our study, daidzein exhibited an inhibitory effect on collageninduced platelet aggregation. Therefore, we investigated the effect of daidzein on granule release in collagen-induced platelets. Figure 2A,B show that when platelets were stimulated with collagen (2.5 µg/mL), ATP and serotonin release increased, but pretreatment with daidzein (12.5-50 µM) significantly and dose-dependently inhibited these releases. Furthermore, daidzein significantly reduced the collagen-induced surface expression of P-selectin ( Figure 2C). These results suggest that daidzein inhibits collagen-induced platelet aggregation by suppressing granule release.

Daidzein Inhibited Thromboxane A 2 Production Regardless of COX-1 Activity in Collagen-Induced Platelets
Platelet aggregation plays a key role in the development of atherosclerosis and thrombosis, and TXA 2 stimulates this process [13]. In our study, the production of TXA 2 was indirectly verified using its stable metabolite TXB 2 . The production of TXB 2 increased when platelets were treated with collagen (2.5 µg/mL), but pretreatment with daidzein (12.5-50 µM) significantly attenuated the production of TXB 2 ( Figure 3A). In platelets, COX-1 is essential for the synthesis of TXA 2 [29]. However, our results show that daidzein (12.5-50 µM) had no inhibitory effect on COX-1 activity ( Figure 3B), whereas the positive controls, aspirin (500 µM) and SC-560 (3.3 µM) (a COX-1 inhibitor), both reduced COX-1 activity. These results suggest that daidzein inhibits TXA 2 production through a mechanism other than COX-1 activity.

Inhibitory Effect of Daidzein on the Phosphorylation of Cytosolic Phospholipase A 2
cPLA 2 releases arachidonic acid, COX-1 converts arachidonic acid into prostaglandin H 2 , and TXA 2 is derived from prostaglandin H 2 , playing a role in platelet aggregation and vasoconstriction [30]. Therefore, we investigated the effect of daidzein on the phosphorylation of cPLA 2 , an upstream signaling pathway that regulates the production of TXB 2 . When platelets were treated with collagen (2.5 µg/mL), cPLA 2 phosphorylation increased ( Figure 3C). However, this increase was attenuated in the presence of daidzein (12.5-50 µM). These results suggest that daidzein inhibits the phosphorylation of cPLA 2 rather than COX-1 activity, thereby inhibiting TXB 2 production in collagen-induced platelet aggregation.

Effects of Daidzein on Cyclic Adenosine Monophosphate Levels and Phosphodiesterase Activity in Collagen-Induced Platelets
cAMP is an important mediator of platelet activity and inhibits platelet aggregation [31]. Thus, we assessed the effects of daidzein on cAMP levels after collagen-induced platelet aggregation. When pretreated with daidzein (12.5-50 µM), cAMP levels significantly and dose-dependently increased versus collagen ( Figure 4A). Furthermore, pretreatment with dipyridamole (20 µM), a phosphodiesterase (PDE) inhibitor and positive control, also increased cAMP levels versus collagen. Platelets contain several PDEs that catalyze the degradation of cAMP, a second messenger that regulates platelet activation [5]. The activity of PDE was indirectly confirmed whether daidzein inhibits the activity of PDE, an enzyme that degrades cAMP, by measuring the level of cAMP in not stimulated with collagen. Figure 4B shows that daidzein (50 µM) and dipyridamole (20 µM) both increased cAMP levels in unstimulated platelets. These results suggest that daidzein increases cAMP levels by inhibiting cAMP hydrolysis by attenuating PDE activity.

Effects of Daidzein on the Phosphorylations of VASP (Ser 157 ) and IP 3 R1 in Collagen-Activated Platelets
Increasing cAMP levels causes protein kinase A (PKA) activation, and activated PKA causes the phosphorylations of VASP and IP 3 R [14]. Therefore, we investigated the effects of daidzein on the phosphorylations of VASP (Ser 157 ) and IP 3 R1. The results showed that the phosphorylations of VASP (Ser 157 ) and IP 3 R1 were reduced by collagen (2.5 µg/mL). However, when platelets were pretreated with daidzein (12.5-50 µM) and then with collagen, the phosphorylations of VASP (Ser 157 ) and IP 3 R1 were increased as compared with collagen-treated platelets ( Figure 4C,D). These results suggest that daidzein increases the phosphorylations of VASP (Ser 157 ) and IP 3 R1 by increasing cAMP levels.

Inhibitory Effect of Daidzein on Clot Retraction
Outside-in signaling through integrin α IIb β 3 plays a critical role in clot retraction due to fibrinogen binding [33]. Therefore, we investigated the effect of daidzein on clot retraction after stimulating platelets with thrombin (0.05 U/mL). Clot retraction was observed when PRP was treated with thrombin (0.05 U/mL), and this was significantly inhibited by daidzein pretreatment (12.5-50 µM) ( Figure 5C,D). These results suggest that daidzein inhibits platelet-induced clot retraction by inhibiting the activation of integrin α IIb β 3 .

Inhibitory Effects of Daidzein on the Phosphorylations of PI3K/PDK1/Akt/GSK3αβ and MAPK (p38 and ERK) Pathways
The PI3K/Akt pathway plays an important role in collagen-induced platelet aggregation as it phosphorylates and activates various targets including proteins involved in granule release and the activation of integrin α IIb β 3 [20,21]. Therefore, we investigated daidzein-mediated phosphorylation changes in the PI3K/PDK1/Akt/GSK3αβ pathways in collagen-induced platelets and found that daidzein pretreatment (12.5-50 µM) significantly suppressed their phosphorylations ( Figure 6A). The MAPK pathway promotes platelet activation and aggregation by regulating processes involved in platelet function such as the activation of integrins and the production of TXA 2 [34]. Our results show that daidzein significantly attenuated the phosphorylations of MAPK (p38, ERK) pathways in collagen-induced platelets ( Figure 6B).

The Synergistic Effect of Daidzein and Aspirin
We found that daidzein inhibited collagen-induced platelet aggregation and thrombininduced clot retraction without affecting the COX-1 pathway. Therefore, we confirmed the synergistic effect when aspirin, a COX inhibitor, was treated together with daidzein in a low concentration. Figure 7A shows that cotreatment with daidzein (12.5 µM) and aspirin (50 µM) strongly inhibited platelet aggregation at these low concentrations, whereas, when treated individually, daidzein and aspirin weakly inhibited platelet aggregation. Furthermore, daidzein and aspirin cotreatment at these low concentrations inhibited the production of TXB 2 in collagen-stimulated platelets ( Figure 7B). These results suggest that the side effects of COX-1 inhibition by aspirin can be reduced by daidzein coadministration.

Discussion
CVD is a group of disorders that includes coronary artery disease, heart failure, arrhythmias, and stroke [35], all of which affect the heart and blood vessels. Platelet aggregation is an important process during the development of CVD. Platelets are small blood cells that play a crucial role in blood clotting, which is essential to stop bleeding after injury. However, under certain circumstances, platelets can become activated and clump together to form blood clots that can block blood vessels [36]. This process is known as platelet aggregation and is a critical component of atherosclerotic plaque formation, which is the primary cause of coronary artery disease. In addition to contributing to the formation of atherosclerotic plaque, platelet aggregation can also result in blood clots in the arteries that supply blood to the heart (coronary thrombosis) and cause heart attacks. Platelet aggregation can also contribute to the development of other types of CVD, including stroke [37,38].
Daidzein is an isoflavone found in soy products and various plants and has been reported to have several health benefits, including anti-inflammatory and antioxidant effects [28]. Furthermore, evidence suggests that daidzein might inhibit platelet activation, although the precise mechanism is not fully understood. In this study, we sought to reveal the mechanism responsible for the anti-platelet effect of daidzein. We found that daidzein had inhibitory effects on collagen-induced platelet aggregation, granule release, TXA 2 production, integrin α IIb β 3 activation, and clot retraction, and that it increased cAMP levels and the phosphorylations of VASP (Ser 157 ) and IP 3 R1 but attenuated the phosphorylations of PI3K/PDK1/Akt/GSK3αβ and MAPKs (p38 and ERK) (Figure 8).
Activation leads to the release of alpha granules, dense granules, and lysosomes by platelets [39]. Alpha granules contain a variety of proteins, such as fibrinogen, von Willebrand factor, and platelet-derived growth factor, which are important for platelet function and wound healing [10]. Dense granules contain small molecules, such as ADP and serotonin, which are released upon platelet activation and contribute to platelet aggregation and blood clot formation [8]. We found that daidzein attenuated the release of granules (ATP, serotonin, and P-selectin), which suggests daidzein inhibits platelet activation.
When platelets are activated, they activate cPLA 2 , which acts on phospholipids in the cell membrane to release AA. This released AA is then converted into prostaglandins and thromboxane by COX enzymes. Furthermore, thromboxane is synthesized in platelets by TXAS and is involved in platelet aggregation and clot formation [13]. Our results show that daidzein did not affect COX-1 but inhibited thromboxane production by suppressing cPLA 2 phosphorylation. Activation leads to the release of alpha granules, dense granules, and lysosomes by platelets [39]. Alpha granules contain a variety of proteins, such as fibrinogen, von Willebrand factor, and platelet-derived growth factor, which are important for platelet function and wound healing [10]. Dense granules contain small molecules, such as ADP and serotonin, which are released upon platelet activation and contribute to platelet aggregation and blood clot formation [8]. We found that daidzein attenuated the release of granules (ATP, serotonin, and P-selectin), which suggests daidzein inhibits platelet activation.
When platelets are activated, they activate cPLA2, which acts on phospholipids in the cell membrane to release AA. This released AA is then converted into prostaglandins and thromboxane by COX enzymes. Furthermore, thromboxane is synthesized in platelets by TXAS and is involved in platelet aggregation and clot formation [13]. Our results show that daidzein did not affect COX-1 but inhibited thromboxane production by suppressing cPLA2 phosphorylation.
cAMP acts as a second messenger and inhibits platelet activation, whereas PDEs are responsible for the breakdown of cAMP to AMP [40]. The present study indirectly demonstrates that daidzein increases cAMP levels by inhibiting PDE activity, which suggests a potential mechanism for its inhibitory effect on platelet activation. Furthermore, in collagen-induced platelets, daidzein increased the phosphorylations of two cAMP-dependent proteins, VASP and IP3R1, which are regulated by cAMP and play key roles in the regulation of platelet function [31].
VASP plays a key role in clot retraction, that is, the process by which blood clots are compacted and strengthened. During clot retraction, platelets change shape and pull on fibrin strands to reduce clot sizes [41]. Integrins are transmembrane proteins involved in platelet adhesion and aggregation and mediate interactions between platelets and the cAMP acts as a second messenger and inhibits platelet activation, whereas PDEs are responsible for the breakdown of cAMP to AMP [40]. The present study indirectly demonstrates that daidzein increases cAMP levels by inhibiting PDE activity, which suggests a potential mechanism for its inhibitory effect on platelet activation. Furthermore, in collagen-induced platelets, daidzein increased the phosphorylations of two cAMPdependent proteins, VASP and IP 3 R1, which are regulated by cAMP and play key roles in the regulation of platelet function [31].
VASP plays a key role in clot retraction, that is, the process by which blood clots are compacted and strengthened. During clot retraction, platelets change shape and pull on fibrin strands to reduce clot sizes [41]. Integrins are transmembrane proteins involved in platelet adhesion and aggregation and mediate interactions between platelets and the extracellular matrix and, thus, play important roles in platelet function and clot formation [16,18]. The regulations of VASP and integrin α IIb β 3 are critical for maintaining hemostasis and preventing thrombotic disorders because they play important roles in platelet function, including clot retraction. Our study shows that daidzein-induced increases in cAMP level and VASP (Ser 157 ) phosphorylation are closely related to the inhibition of integrin α IIb β 3 activation. Furthermore, our findings indicate that the inhibitory effect of daidzein on integrin activity has an impact on clot retraction.
The PI3K/Akt and MAPK pathways are two important signaling pathways involved in platelet activation and aggregation. The PI3K/Akt pathway is activated in response to various platelet agonists, including thrombin, collagen, and ADP, and promotes platelet activation, granule release, and integrin activation [32,42]. The MAPK pathway is also activated in response to various platelet agonists and is composed of three main compo-nents, namely, ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK. When activated, this pathway promotes platelet activation and aggregation by regulating the expression of genes involved in platelet function, such as integrins and TXA 2 [43,44]. We observed that daidzein suppressed the phosphorylations of PI3K, PDK1, Akt (Ser 473 and Thr 308 ), GSK3αβ, p38, and ERK in collagen-induced platelets, which suggests that the anti-platelet effect of daidzein might be due to the inhibition of integrin α IIb β 3 activation, TXA 2 production, and granule release, which are regulated by these pathways.
Aspirin is an anti-platelet drug and COX inhibitor. However, because it inhibits COX-1 and COX-2, aspirin can cause side effects due to the suppression of COX-1. Currently, numerous studies are being conducted to minimize the adverse effects associated with COX-1 inhibition [45]. Interestingly, we found that daidzein had no effect on COX-1 activity and observed a synergistic effect on platelet aggregation and TXA 2 production when daidzein and aspirin were cotreated at low concentrations. Notably, our results suggest that daidzein may be used to reduce aspirin dosages, potentially decreasing the severity and incidence of side effects caused by COX-1 inhibition.

Preparation of Washed Human Platelets
Human platelet-rich plasma (PRP) was obtained from the Korean Red Cross Blood Center (Daegu, South Korea), centrifuged at 125× g for 10 min to remove unwanted cells, and then centrifuged at 1300× g for 10 min to obtain platelet pellets. Platelet pellets were then washed twice with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO 3 , 0.36 mM NaH 2 PO 4 , 5.5 mM glucose, and 1 mM Na 2 EDTA, pH 6.5) and resuspended in suspension buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO 3 , 0.36 mM NaH 2 PO 4 , 0.49 mM MgCl 2 , 5.5 mM glucose, 0.25% gelatin, pH 6.9). The above procedures were performed at room temperature (RT) to avoid platelet activation by low temperatures.

Determination of ATP and Serotonin Release
After platelet aggregation was stopped by adding ice-cold 5 mM EDTA, the supernatant was centrifuged at 2000× g for 5 min at 4 • C. Released ATP and serotonin levels were assessed using an ATP assay kit (Biomedical Research Service Center, Buffalo, NY, USA) and a serotonin ELISA kit (Abnova, Taipei, Taiwan) using a SpectraMax M2e microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Determination of TXA 2 Production
After platelet aggregation was stopped by adding ice-cold 5 mM EDTA, the supernatant was obtained by centrifugation at 2000× g. It was then diluted 1:500 and used to assess TXA 2 production. Since TXB 2 is a stable metabolite of TXA 2 , TXA 2 production was assessed using a TXB 2 EISA kit (Cayman Chemical, Ann Arbor, MI, USA).

Determination of cAMP Levels
Platelet suspensions (10 8 cells/mL) were preincubated with various concentrations of daidzein or 0.2% dimethyl sulfoxide (DMSO) for 2 min in the presence of 2 mM CaCl 2 and then stimulated with collagen (2.5 µg/mL) for 5 min. Ethanol (750 µL, 80%) was then added to prevent platelet aggregation, and samples were reacted for 30 min at RT then separated by centrifugation at 2000× g for 10 min at 4 • C. Supernatants were then transferred to new tubes and dried by vacuum centrifugation. The dried pellets obtained were dissolved in ELISA buffer from a cyclic AMP ELISA kit (Cayman Chemical, Ann Arbor, MI, USA), and cAMP levels in samples were determined using the SpectraMax M2e (Molecular Devices, Sunnyvale, CA, USA).

Flow Cytometry Analysis
Washed human platelets were preincubated with various concentrations of daidzein (12.5-50 µM) or 0.2% DMSO for 2 min in the presence of 2 mM CaCl 2 and stimulated with collagen (2.5 µg/mL) for 5 min. Samples were then fixed with 0.5% paraformaldehyde for 30 min at 4 • C, washed three times with phosphate-buffered saline (PBS), suspended in ice-cold 3% bovine serum albumin (BSA)/PBS, and incubated with Alexa Fluor 488 antihuman CD62P (P-selectin) antibody or Alexa Fluor 488-conjugated human fibrinogen in 3% BSA/PBS for 30 min at 4 • C in the dark. After centrifugation and washing, platelet pellets were dissolved in 3% BSA/PBS. Flow cytometry was performed using a FACS Calibur II (BD Biosciences, San Jose, CA, USA) and CellQuest version 5.2.1.

Clot Retraction Assay
Human PRP was preincubated with various concentrations of daidzein (12.5-50 µM) or 0.2% DMSO for 5 min at 37 • C and induced to coagulate with thrombin (0.8 U/mL). Clots were allowed to retract numerous times at 37 • C and photographed. Quantification was carried out by measuring clot areas using Image J 1.8.0 software (National Institute of Mental Health, Bethesda, MD, USA). Percentage clot retraction was calculated using: Retraction (%) = 100 − [(sample clot area/intact sample area) × 100] (2)

Immunoblotting Analysis
After platelet aggregation, the reaction was stopped by adding protease and phosphatase inhibitor-contained RIPA lysis buffer (50 mM pH 7.5 Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 2 mM EDTA). Protein concentrations were determined using a BCA assay. Equal amounts of proteins from platelet lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes, which were then blocked with 5% skim milk in TBS buffer. Membranes were reacted with primary antibodies overnight at 4 • C and with secondary antibodies for 2 h at RT. Protein bands were visualized using a chemiluminescent substrate and photographed using a LAS-4000 (Fujifilm, Tokyo, Japan) luminescent image analyzer.

Statistical Analysis
Analysis of variance (ANOVA) was used to determine the significances of intergroup differences, and when ANOVA indicated a significant difference, groups were further compared using Tukey's post hoc test in SPSS V20.0 software (SPSS, Inc., Chicago, IL, USA). Results are presented as means ± standard deviations and numbers of observations. p values of <0.05 were considered statistically significant.

Conclusions
Our study indicates that daidzein inhibits collagen-induced platelet aggregation by activating cAMP and inhibiting TXA 2 production and the PI3K/Akt and MAPK p38/ERK signaling pathways. This inhibition of platelet activation by daidzein reduces granule release and fibrinogen binding to integrin α IIb β 3 and, ultimately, inhibits platelet-mediated clot retraction. Taken together, these findings suggest that daidzein may have therapeutic potential as an anti-platelet and anti-thrombotic agent. However, further research is needed to determine the optimal dose and potential side effects of daidzein as a therapeutic agent and its efficacy and safety in clinical settings.  Informed Consent Statement: Blood samples are provided for research using blood from unspecified blood donors. The Red Cross Blood Center undergoes its own research ethics review, and, at this time, the research is conducted with the approval of an exemption from obtaining the blood donor's written consent.

Data Availability Statement:
The public available databases analyzed during the current study are included in the article, further inquiries can be directed to the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.